JP2680713B2 - Method for counting live microbial cells - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for measuring living microbial cells, and in particular, living cells applied to quality control and sterilization performance evaluation of raw materials and products in food plants and pharmaceutical manufacturing plants. The present invention relates to a counting method.

[Conventional technology]

In food manufacturing and pharmaceutical manufacturing plants, microbial inspection and measurement are performed for quality control of raw materials and products, and sterilization performance evaluation, and a great deal of labor and time are spent on this. Conventionally, the agar culture method has been most widely used as a microorganism inspection method.This method involves dispersing and culturing a sample on agar in which a nutrient source of a microorganism is dissolved, and forming a colony on the agar. The number is counted to measure living cells. However, since this method involves a culture operation, it usually requires a long inspection time of 1 to several days, and quality control,
In many cases, this will interfere with sterilization management.

[Problems to be solved by the invention]

The biggest problem with the conventional method is that it involves a culture operation,
It requires a long time for inspection and measurement.

The present invention is intended to solve the problems of the above-mentioned conventional methods and provide a method capable of measuring live cells with high sensitivity in a short time.

Therefore, as a result of various studies on a method for detecting live bacteria without requiring a special culture operation, fluorescein diacetate (hereinafter, abbreviated as FDA), which is a kind of fluorescein derivative, is allowed to act on a sample under special conditions. Therefore, we have found a method for detecting viable bacteria in a short time with high sensitivity.

It has been known that FDA reacts with esterase, which is a type of enzyme, to produce fluorescein, which is a fluorescent substance.Using this property, FDA acts on animal cells to produce fluorescein produced intracellularly. There are examples in which the fluidity of the cytoplasm is obtained from the degree of polarization and is used for diagnosis, and FDA is used for confirmation of plant cell protoplast cell wall regeneration. However, it has been rarely applied to the measurement of living cells of microbial cells, and little is known about the FDA usage conditions and the effectiveness of FDA.

The present inventors, in order to investigate the possibility of measuring living microbial cells using FDA, as a result of conducting experiments on FDA usage conditions and fluorescence emission characteristics, when FDA is allowed to act on a sample, fluorescence generated in cells I learned that there is a problem that the substance (fluorescein) gradually flows out of the cell, the background fluorescence becomes high, and the difference between the cell fluorescence and the background fluorescence becomes small, and accurate measurement cannot be performed.

The present invention intends to solve this problem and provide a method for accurately measuring living cells.

[Means for solving the problem]

The first invention of the present invention is: (1) The fluorescein derivative is allowed to act on a sample to be measured to excite a fluorescent substance accumulated in living cells to detect fluorescence emitted from the living cells, thereby determining the number of living microorganisms. In the method of measurement, a fluorescein derivative dissolved in a solvent is added to a sample, and the sample is kept at a constant temperature for a predetermined period of time, and then acid is applied to lower the pH, and then the sample is irradiated with excitation light to emit cells. A method for counting live microbial cells, which comprises detecting fluorescence. (Hereinafter, referred to as the first invention) In the first invention, the fluorescein derivative dissolved in the solvent is generally further diluted several tens of times with a phosphate buffer and allowed to act on the sample at a predetermined concentration. If this is done, the FDA will deteriorate significantly, and there will be a problem that the fluorescence emission amount of the cells will decrease in a short time with the reagent that has been prepared for 1-2 hours. Second solution of the present invention as a solution
The invention has the following configuration.

(2) In the method for measuring the number of living cells of a microorganism, the fluorescein derivative is allowed to act on the sample to be measured, and the fluorescent substance accumulated in the living cell is excited to detect the fluorescence emitted from the living cell. After adding a fluorescein derivative that is not diluted with phosphate buffer to the sample and holding it at a constant temperature for a certain period of time, acid is applied to lower the pH, and then the sample is irradiated with excitation light to detect the fluorescence emitted by cells. A method for counting viable microbial cells, which comprises detecting. (Hereinafter, referred to as second invention) As the fluorescein derivative used in the present invention, in addition to FDA, fluorescein-sodium (uranin) and fluorescein- (β-D-galactophyranoside) can be used.

[Operation of the First Invention] Like living and plant cells, it is considered that the enzyme esterase is present in living cells of microbial living cells, and the enzyme is not present in dead cells because the enzyme is inactivated. Therefore, when FDA acts on a microbial sample, only living cells react with FDA to produce fluorescein in the cells. At this time, conditions such as temperature and time are essential to promote the reaction. The optimum temperature is 35-37 ℃, 10 ℃ or less, or 45 ℃
With the above, no reaction occurs.

In addition, for the purpose of rapid measurement, a shorter reaction time is desirable, but it is necessary to generate a fluorescent substance that can be measured in cells, and the reaction time for this is, in the case of yeast,
It takes 5 to 10 minutes, and 10 to 20 minutes for bacteria such as Escherichia coli and Bacillus subtilis and spores such as mold.

Therefore, when FDA is allowed to interact with the sample for a certain period of time, a fluorescent substance is produced inside the cell, but with time, the fluorescent substance produced inside the cell also begins to flow out of the cell, and the fluorescent emission amount of living cells is increased. It was found that there is a defect that the difference between the fluorescence emission amount and the background becomes small and measurement becomes difficult. Therefore, as a result of various studies on this countermeasure, the background fluorescence emission is extinguished by adding an acid to the sample and adjusting the pH to 4 or less. Since cells have protective functions such as cell walls and cell membranes, it took time for the acid to enter the cells, and it was found that the fluorescence emission of the cells did not disappear.

According to the method of the first aspect of the present invention, the difference in the fluorescence emission amount between the live cells and the background becomes large, and the live cells can be measured with high sensitivity.

To excite the fluorescent substance produced in living cells,
It is necessary to irradiate light having a specific wavelength range, but this excitation spectrum is a relatively broad curve having a peak near 490 nm as shown in FIG. When excited at a wavelength of 490 nm, the intracellular fluorescent substance emits a fluorescence spectrum having a peak in the vicinity of 510 to 515 nm as shown in FIG.

Therefore, by irradiating with light having a wavelength required to excite the fluorescent substance generated in such cells, individual cells emit fluorescence, and by measuring this as a point of light, living cells are It is possible to measure with high accuracy.

[Operation of the Second Invention] Once FDA dissolved in acetone is diluted with a phosphate buffer solution, the FDA is easily altered and decomposed,
Experiments have shown that the effect decreases rapidly. Therefore, unlike the method that was conventionally used for plant cells, etc., FDA dissolved in acetone is directly dissolved in acetone as a result of directly acting on the microorganism sample without diluting FDA dissolved in acetone with a phosphate buffer solution. After that, it was found that it could be stably maintained even after 10 hours or more, and a great effect was obtained on living microbial cells.

〔Example〕

 The configuration of the living cell measuring device used in the test is shown in FIG.

In FIG. 1, 1 is an excitation light source, a mercury lamp with an output of 100 W, 2 is a collector lens for collecting the mercury light, 3 is an excitation filter, which is necessary to excite the fluorescent substance generated in living cells It is a filter that passes only the wavelength. In this experiment, an excitation filter that passes the wavelength region of 450 to 490 nm was used.

Reference numeral 4 is a mirror, and 5 is an objective lens having a magnification of 20 times. A microbial sample, which has been lowered in pH by reacting with FDA, is dropped on a slide glass, covered with a cover glass, placed on the sample table 6, and irradiated with excitation light to remove viable cells.
It emits fluorescence whose main wavelength is 510 to 515 nm. Reference numeral 7 denotes an absorption filter which allows light having a wavelength of 510 nm or more to pass therethrough, and the television camera 9 images the luminescent cells through the lens 8. Reference numeral 10 denotes an image processing apparatus capable of outputting an image of a luminescent cell present within an arbitrarily set luminance level. The image processed image is displayed on a monitor 12. A particle counter 11 measures the number of cells in an arbitrary brightness range. 13 is a data processing analysis device, which is the number of cells in a predetermined brightness range,
The brightness distribution of cells is output, and the number of viable cells can be accurately determined.

 Next, a test example using the above apparatus will be shown.

(1) Cells used in the test Spores were collected from black mold (Aspergillus nigar) cultured on potato dextrose agar for about 1 week and suspended in physiological saline (pH 7.0) at a concentration of about 10 ⁷ cells / ml. The sample was used.

(2) FDA solution FDA was dissolved in acetone to a concentration of 1 mg / ml.

(3) Working temperature, pH Temperature and pH were set at 37 ° C. and 7.0, 1 ml of cell suspension was taken in a test tube, and FDA solution was added, and then the reaction was carried out for a certain period of time.

(4) Experiment A solution prepared by dissolving FDA in acetone to a concentration of 1 mg / ml was
Diluted 5 times with H7.0 phosphate buffer and added at a ratio of 1: 1 (0.1 mg / ml as FDA concentration) to the cell suspension, and directly to the cell suspension without dilution With respect to the sample added so as to have a concentration of 0.1 mg / ml, the reaction was performed at predetermined intervals for 20 minutes to measure the brightness of the cells.

FIG. 4 shows the relationship between the average brightness of cells and time. As can be seen, the cell brightness of the one diluted with phosphate buffer decreases rapidly with the passage of time.
It can be seen that when the acetone solution in which A is dissolved is added directly, stable light emission can be obtained for a long time.

This means that according to the second aspect of the invention, reliable data can be obtained without frequently changing the FDA solution, which is extremely convenient in terms of work efficiency.

Next, FDA-dissolved acetone solution was directly added to 1 ml of cell suspension to a concentration of 0.1 mg / ml, and the reaction time was changed to measure the cell brightness and background. Show. The abscissa represents the luminescence amount (brightness) of cells, and the ordinate represents the ratio of the measured luminescent cell number to the total cell number No within a predetermined brightness range.

From these results, the luminescence of cells was at a low level overall at a reaction time of 5 minutes, and about 40% of the cells below the background were present, but the luminescence quantity became high at a reaction time of 10 minutes. However, about 10% is still not measured below the background.

As the reaction time increases to 20 minutes and 30 minutes, the fluorescent substances produced inside the cells flow out to the outside of the cell, the background value increases, and the number of cells below the background is 25% of the total, It will increase to 40%.

FIG. 6 shows the method of the present invention. Similarly, a certain amount of a sample was taken out from the test tube at each time, the pH was adjusted to 3 to 4 with 1N hydrochloric acid, and then the number of luminescent cells was measured. As is clear from these results, the background value decreased under all conditions when the reaction time was 10 minutes or more, and the total cell number was 100%.
You can see that% is measured. However, in the method of the present invention, if a long time is added after the addition of the acid, the added acid will penetrate into the cells and inhibit the emission of the fluorescent substance in the cells, so caution is required. The method of the present invention is applicable to yeast, Bacillus subtilis spores, and Escherichia coli as well as black mold spores.

〔The invention's effect〕

In the conventional method, the measurement time of living microbial cells required a long time of 1 to several days, but in the method of the present invention, it is significantly quicker with about 10 minutes, and since it can be measured with high sensitivity, foods, raw materials in the pharmaceutical field, It enables rapid quality control of products and evaluation of sterilization performance, and has a great effect on reduction of production cost.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of an apparatus used for carrying out an embodiment of the present invention, FIG. 2 is a graph of an excitation wavelength spectrum required for fluorescence emission of living cells, and FIG. 3 is a graph of a fluorescence spectrum emitted by living cells. FIG. 4 is a graph showing a change in cell brightness with the passage of time since the FDA solution was prepared according to the example of the present invention, FIG. 5 is a graph showing the relationship between the brightness and the cell number ratio according to the conventional method, and FIG. 3 is a graph showing the relationship between the brightness and the cell number ratio according to the embodiment of the present invention.

Claims (2)

(57) [Claims] 1. A method for measuring the number of viable cells of a microorganism by allowing a fluorescein derivative to act on a sample to be measured, exciting a fluorescent substance that accumulates in the viable cell, and detecting fluorescence emitted from the viable cell. The fluorescein derivative was added to the sample, kept at a constant temperature for a fixed period of time, and then acid was applied to lower the pH, and then the sample was irradiated with excitation light to detect fluorescence emitted by cells. A method for counting living microbial cells. 2. A method for measuring the number of viable cells of a microorganism by allowing a fluorescein derivative to act on a sample to be measured, exciting a fluorescent substance that accumulates in the viable cell, and detecting fluorescence emitted from the viable cell. Add fluorescein derivative not diluted with phosphate buffer to the sample,
A method for counting viable microbial cells, which comprises holding a certain temperature and a certain period of time, then applying an acid to lower the pH, irradiating the sample with excitation light, and detecting the fluorescence emitted by the cells.